PROJECT SUMMARY Dravet syndrome is a severe neurodevelopmental disorder that affects 1 in 16,000 children and is defined by treatment-resistant epilepsy, developmental delay, intellectual disability, autism spectrum disorder, and a high rate of sudden death. Dravet syndrome is caused by mutation in the gene SCN1A, which encodes the sodium (Na+) channel Nav1.1 How SCN1A mutation leads to the clinical entity known as Dravet syndrome remains unclear; this gap in knowledge has profoundly limited the practical impact that such a diagnosis has on treatment, quality of life, and long-term outcome for patients with this disorder. Prior work in experimental animal models of Dravet syndrome including Scn1a+/- mice suggests that loss of Nav1.1 leads to epilepsy via dysfunction of GABAergic inhibitory interneurons in the cerebral cortex, with the most prominent identified abnormalities being impaired action potential generation in a critical subtype of interneuron known as the parvalbumin-positive fast-spiking interneuron (PV-IN). However, data presented here indicates that, surprisingly, PV-IN dysfunction is transient, being restricted to a brief time window in early development, with subsequent recovery of high frequency firing. Preliminary data suggests that the specific locus of pathology in Dravet syndrome is actually PV-IN axons, with abnormal action potential propagation leading to conduction delay and synaptic failure, even though PV-INs have recovered the ability to generate action potentials at high frequency. This finding has important implications for the development of novel treatment approaches for Dravet syndrome, such as cell transplantation, gene therapy, or precision medicine. This new 5-year application from the lab of an early stage investigator uses innovative neuroscience approaches to test this new hypothesis as to the mechanism of pathology in Dravet syndrome. Proposed experiments will establish the molecular identity and physiological properties of Na+ channels in PV-IN axons in Scn1a+/- mice as compared to wild-type controls using targeted recordings from interneuron axons and detailed immunohistochemistry of axonal Na+ channels (Aim 1); determine the impact of PV-IN axonal dysfunction on the timing of feedforward inhibition in cerebral cortical circuits (Aim 2); and assess the activity of defined subsets of neurons in awake, behaving Scn1a+/- mice using in vivo imaging and electrophysiology to corroborate in vitro findings (Aim 3). The overall outcome of the proposed experiments will set forth a unifying hypothesis as to the pathophysiology of Dravet syndrome. Such knowledge is critical to the development of novel, targeted therapies for this currently incurable and untreatable disease. The long-term objective of this line of research is to apply preclinical data from experimental model systems to the development of new, mechanistically oriented therapies in human patients.